Leptin is an adipocyte hormone that functions as the afferent signal in a negative feedback loop that regulates adipose tissue mass. As a key element of this feedback loop, leptin both responds to changes in adipose tissue mass and in turn modulates the size of the adipose depot. This application proposes a set of experiments that seek to elucidate the molecular mechanisms that are responsible for this reciprocal relationship between leptin and adipose tissue mass. In the first set of experiments, we propose elucidate the molecular mechanisms that control leptin gene expression. Leptin plasma and RNA levels can vary several hundred fold between the fasted and obese states and the transcriptional mechanisms responsible for its adipose tissue specific expression and this quantitative regulation are not known. Leptin is expressed at significantly higher levels in vivo vs. in vitro making it necessary to study leptin gene expression using transgenic mice. We have used an in vivo luciferase imaging system to localize the cis elements necessary for tissue specific and quantitative regulation to between -22 kB and +18 kB of the leptin gene. We propose further experiments to test a set of promoter deletions to map the cis elements and trans factors regulating leptin expression as a prelude to defining the relevant signal transduction pathway. We hypothesize that leptin is regulated by a lipid sensing system and, if true, these experiments could lead us to understand how intracellular lipid content is sensed and read out by the leptin gene, In the second set of experiments, we will explore the mechanism by which changing leptin levels control adipose tissue mass. While leptin deficient obese mice show a massive increase in the number of fat cells, the factors regulating fat cell production are largely unknown. We have recently identified an adipocyte stem cell that is capable of reconstituting a fat depot and correcting the metabolic abnormalities of fat deficient lipodystrophic mice. We now propose to further characterize this cell type in vivo and in vitro as a prelude to studies of the effects of leptin on the growth and development of this novel cell type. We also propose to study adipose tissue development by employing an ES cell complementation method that we have developed. In this method, wild type ES cells are injected into blastocysts of lipodystrophic animals. In the resulting chimaeras, the ES cells are the sole source of adipose tissue. This technique will allow us to titrate the number of ES cells to define the minimal clone size required for development of the adipose mass, results which will have important implications for our understanding of the ability of adipose tissue precursors to reconstitute adipose tissue. This method also provides a robust and efficient means for studying the role of specific gene products that will be identified in the other experiments in regulating adipose tissue development and function. PUBLIC HEALTH RELEVANCE: Obesity is associated with Type II diabetes, hypertension, hyperlipidemia and hepatic steatosis and represents a major public health problem (1). Leptin, an adipocyte hormone, regulates adipose tissue mass as part of a feedback loop and a fuller understanding of the elements of this system could have important implications for the pathophysiology and treatment of obesity. In this application, we propose to address two unanswered questions in leptin physiology. What controls the amount of leptin that is produced in the lean vs. obese state? How do changes in leptin concentration in turn regulate adipose tissue mass?